The present invention relates to methods of determining the concentration of a selected drug in the body of a subject to provide the monitoring of either drug levels in a clinical setting and in public health services and patient compliance with medication prescriptions. The methods are characterized by a simplified and effective deproteinizing body fluid step followed by drug extraction and measurement using an accurate technique, such as a colorimetric assay or a High-Performance Liquid Chromatography method.
In the field of medicine, a number of medications have been found safe and efficacious for the treatment of patients with physical illnesses. Patients placed on prescribed medication treatment plans are typically monitored. Subjective and objective methods are used to identity bothersome symptoms and to implement any changes necessary during the course of treatment. Monitoring may continue for as long as treatment is provided.
Currently, the most common method of monitoring patients for medication compliance is clinical observation which involves individual counseling and close personal supervision by physicians which observe physiological signs and symptoms or residual signs of illness and also listen to patient complaints regarding degree of pain relief and evaluate physiological changes over time. This method is time consuming, expensive and highly subjective. Needless to say, it is fraught with potential errors.
Additional compliance information can also be obtained using qualitative urine monitoring methods such as the standard laboratory procedure called enzyme-multiplied immunoassay (EMIT). Utilizing an arbitrary cutoff value, these methods provide the clinician with a simple positive or negative indication of the possible presence or absence of a parent drug or its metabolites in a patient""s urine. The parent drug is the prescribed medication itself and the metabolites are those chemical derivatives of the medication which naturally occur upon the patient""s body metabolizing the medication. These tests do not provide information concerning the time or amount of last drug use or whether or not the prescribed dose of medication was ingested properly, diverted or supplemented.
Physicians utilizing only clinical evaluation and qualitative urine drug screening test results may develop problems in their treatment methods. Consistently, Fox, W. (Fox, W. (1990). xe2x80x9cDrug combinations and bioavailability of rifampicinxe2x80x9d. Tubercle. 71: 241-5) suggested parallel serum/plasma sampling in selected studies for testing abroad to verify the tuberculosis treatment effectiveness using drug combinations by confirming the urinary bioavailability determination. In the mentioned text, the term xe2x80x9cabroadxe2x80x9d means outside developing countries in which expensive analytical equipment is not commonly found.
Another monitoring method sometimes used is a direct measurement of parent drug concentrations or active metabolites concentrations of the drug in plasma and other body fluids. This direct method presents some limitations since it is expensive and requires the use of time consuming and highly technical analytical procedures such as high-performance liquid chromatography and mass spectrometry since active and inactive metabolites must be quantified separately.
Attempts have been made to overcome the difficulties of the sophisticated analytical procedures. In the EP 122 032, it is described a method of determining the concentration of a selected drug in the body of a subject consisting of the steps of holding a liquid collecting means comprising an absorbent inert member, containing a reagent substance which reacts with selected drug, in a position in close proximity to an eye of the patient for collecting tear fluid therefrom and allowing the tear fluid collected to come into contact with said reagent substance during a period sufficient to permit the development of the reaction which has to be physically detectable. It is mentioned that this method provides a readily indication of she level of said drug in the body because the tear fluid is less complex then other body fluids such as blood. Nevertheless, this assay permits only qualitative or semi-quantitative drug detection.
Although simplicity is an important quality when dealing with monitoring methods, the accuracy of the assay is crucial in the control of diseases, e.g. tuberculosis, specially to measure small quantities of drugs in complex body fluids, such as blood. In the U.S. Pat. No. 4,656,141 it is proposed a high-performance liquid chromatography process for detecting the presence of trace amounts of non-fluorescent soluble compounds each having at least one labile hydrogen atom in a carrier solution by adding a non-fluorescent quinone which is reducible to a fluorescent hydroquinone, and irradiating the resulting solution in the absence of oxygen with light of sufficient energy to cause the quinone to be reduced to a hydroquinone.
Preferably both quantitative and analytical methods should be used to follow the patient on a repetitive basis to ensure that the patient is indeed ingesting the prescribed amounts of medication in the proper manner and responding as expected. Moreover, in control programmes of Public Health Services, confident monitoring of treatment is crucial. Tuberculosis Control Program may be cited as a representative example of this approach and Rifampicin as a highly potent drug widely used for tuberculosis treatment.
An efficient follow up the other drugs treatment performance is also important. Examples are anti-retroviral drugs, such as proteinase or reverse transcriptase inhibitors, e.g. 2xe2x80x2,3xe2x80x2-dideoxyinosine (ddI), 2xe2x80x2,3xe2x80x2-dideoxycytidine (ddC) or 3xe2x80x2-azido-2,3xe2x80x2-dideoxythymidine (AZT) (see Frijus-Plessen N., Michaelis H. C., Forth H. e Kahl G. F. (1990). xe2x80x9cDetermination of 3xe2x80x2-azido-3xe2x80x2-deoxythymidine, 2xe2x80x2,3xe2x80x2-dideoxycytidine, 3xe2x80x2-fluoro-3xe2x80x2-deoxythymidine and 2xe2x80x2,3xe2x80x2-dideoxyinosine in biological samples by high-performance liquid chromatographyxe2x80x9d. Chrombio. Elsevier Science Publishers B. V. Amsterdam. 534: 101-107), anti-fungal drugs, e.g. itraconazole which is also used in anti-leishmanial chemotherapy. (Anon: British Society for Antimicrobial Chemotherapy Working Party: Laboratory monitoring of antifungal chemotherapy. The Lancet. Vol. 337. pp. 1577-1580. 1991) or antimonials, the most used anti-leishmanial drug (World Health Organization. Tropical Disease Research. Twelfth Programme Report. World Health Organization Geneva, Switzerland. Pp 139.1995).
In the case of patients with tuberculosis, there has been increasing interest in the determination of serum levels of the main antituberculosis drugs, in particular the most used rifampicin medication. The usual methods for rifampicin assay are colorimetry, microbiology and high-performance liquid chromatography. In the beginning, microbiological assays were employed by using Sarcina lutea or Staphylococcus aureus. Examples are described in: Furesz S., Scotti, P., Pallanza R., Mapelli E. (1967). xe2x80x9cRifampicin: A new, rifampicin. III Absorption, distribution and elimination in manxe2x80x9d. Arzneim-Forsch. 17: 534-7; Boman, G. (1974). xe2x80x9cSerum concentration and half-life of rifampicin after simultaneous oral administration of aminosalicylic acid or isoniazidxe2x80x9d. Europ J Clin Pharmacol. 7: 217-25; Dickinson, J. M., Aber, V. R., Allen, B. W., Ellard, A., Mitchison, D. A. (1974). xe2x80x9cAssay of rifampicin in serumxe2x80x9d. J Clin Path. 27: 457-62; Buniva, G., Pagani, V., Carozzi, A. (1983). xe2x80x9cBioavailability of rifampicin capsulesxe2x80x9d. Int J Clin Pharmacol Therapy Toxicol. 21: 404-9; Immanuel, C., Jayasankar, K., Narayana, A. S. L., Saema, G. R. (1985). xe2x80x9cSelf-induction of rifampicin metabolism in manxe2x80x9d. Indian Med Res. 82: 381-7. However, the precision of such methods is generally poorer than would be expected with HPLC methods.
Colorimetric methods are interesting under the point of view of easier accomplishing. The procedures or such methods are described in: Maggi, N., Furesz, S., Pallanza, R., Pelizza G. (1969). xe2x80x9cRifampicin desacetylation in the human organismxe2x80x9d. Arzneim-Forsch. 19: 651-4; Sunahara, S. , Nakagawa, H. (1972). xe2x80x9cMetabolic study and controlled clinical trials of rifampicinxe2x80x9d. Chest. 61: 526-32; Jeanes, C. W. L., Jessamine, A. G., Eidus, L. (1972). xe2x80x9cTreatment of chronic drug-resistant pulmonary tuberculosis with rifampicin and ethambutolxe2x80x9d. Canad Med Ass J. 106: 884-8; Brechbuhler, S., Flueher, H., Riess, W. (1978). xe2x80x9cThe renal elimination of rifampicin as a function of the oral dosexe2x80x9d. Arzneim-Forsch. 26: 480-3; McConnell, J. B., Smith, H., Davis, M., Williams, R. (1979). xe2x80x9cPlasma rifampicin assay for an improved solvent extraction techniquexe2x80x9d. Br J Clin Pharmc. 8: 506-7; Israili, Z. H., Rogers C. M., El-Attar, H. (1987). xe2x80x9cPharmacokinetics of antituberculosis drugs in patientsxe2x80x9d. J Clin Pharmacol. 27: 78-83
High-Performance Liquid Chromatography (HPLC) has been used for separate determination of rifampicin and its metabolites. HPLC procedures are described in: Goucher, C. R., Peters, J. H., Gordon, G. R., Murray, J. F., Ichikawa, W., Welch, T. M., Gelber, R. H. (1977) xe2x80x9cChemical and bacteriological assays of rifampicin, rifampicin-quinone and desacetylrifampicinxe2x80x9d. 12th U.S.-Japan Joint Conference on Leprosy. Boston. Mass. Sep. 27-29, 1977. pp. 47-59; Lecaillon, J. B., Febvre, N., Metayer, J. P., Souppart, C. (1978). xe2x80x9cQuantitative assay or rifampicin and three of its metabolites in human plasma, urine and saliva by high-performance liquid chromatographyxe2x80x9d. J Chromatogr. 145: 319-24; Ratti, B., Kosina-Parenti, R., Toseili A., Zerrili, L. F. (1981). xe2x80x9cQuantitative assay of rifampicin and its metabolite 25 desacetyl-rifampicin in human plasma by reversed-phase high-performance liquid chromatographyxe2x80x9d. J Chromatogr. 225: 526-31; Guillaumant, M., Leclercq, M., Forbert, Y., Guise, B., Harf, R. (1982). xe2x80x9cDetermination of rifampicin, desacetylrifampicin, isoniazid and acetylisoniazid by high performance liquid chromatography: Application to human serum extracts, polymorphonucieocytes and alveolar macrophagesxe2x80x9d. J Chromatogr. 232: 369-76; Acocella, G., Nonis, A., Gialdroni-Grassi, G., Grassi, C. (1988). xe2x80x9cComparative bioavailability of isoniazid, rifampicin, and pyrazinamide administered in free combination and in a fixed triple formulation designed for daily use in antituberculosis chemotherapyxe2x80x9d. Am Rev Respir Dis. 138: 882-5; Ishii, M., Agata, H. (1988) xe2x80x9cDetermination of rifampicin and its main metabolites in human plasmaxe2x80x9d. J Chromatogr. 426: 412-6; Nau, R., Prange, W. H., Menck, S., Kolenda, H., Visser, K., Seydel, J. K. (1992). xe2x80x9cPenetration of rifampicin into the cerebrospinal fluid of adults with uninflamed meningesxe2x80x9d. J Antimicrob Chemother. 29: 719-24; Chouchane, N., Barre, J., Toumi, A., Tillement, J. P., Benakis, A. (1995). xe2x80x9cBioequivalence study of two pharmaceutical forms of rifampicin capsules in manxe2x80x9d. Eur J Drug Metab Pharmacokin. 20: 315-20.
While providing useful information relative to patient status and treatment compliance, the clinical monitoring methods described above, i. e. clinical interviews with patients, direct plasma drug measurement and qualitative urine drug screening, have distinct drawbacks which limit their usefulness in extended treatment programmes. Although being effective, the complex assays with many extraction steps, e.g. HPLC, require expensive equipment and specialized operating personel and materials which are not easily found in small hospital centers or field laboratories, mainly in developing countries. Moreover, the occurence of losses during the extraction steps lead to lower drug concentrations, and consequently to wrong results.
Thus, it remains a need for methods of monitoring patient compliance whithout the above mentioned disadvantages of the known methods but having sensitivity and specificity sufficient to detect trace amounts of substances contained in complex body fluids. Such monitoring methods would help physicians both in prescribing adequate doses of medication and in monitoring patients to insure that they are ingesting the prescribed amounts. Accordingly, it is to the provision of such improved methods that the present invention is directed.
The object of the invention is to provide the monitoring or either drug levels in a clinical setting and in public health services and patient compliance with medication prescriptions. The drug levels monitoring is, accomplished by quantitative assays which allow drug detection in body fluids down to 0.3 xcexcg/ml. The method based on extraction of the drug from biological fluids is characterized by a prior deproteinizing step in conditions that at least 97% of the drug is recovered, i.e. by carrying out the deproteinizaing step in the presence of ZnSO4 it is possible to efficiently strip off the drug which became bound to proteins contained in the biological fluid. Noteworthy the method of the invention is specially useful for a drug assay from blood which contains much more protein than other biological fluids such as urine, saliva, tear fluid.
One embodiment of the present invention is a method for drug level detection by using a simplified and effective deproteinizing step from body fluids, such as plasma, blood, urine, saliva, tear fluid, followed by drug extraction and measurement using an accurate technique, such as a colorimetric assay or a High-Performance Liquid Chromatography method.
In a particular embodiment, the invention is directed to a method to quantify rifampicin in order to monitor its levels in body fluids and also to a kit of tuberculosis diagnosis based oh rifampicin concentration measurement.